This revised application is a request for renewal of R01 DA020686. A major accomplishment of the previous funding period was identifying a key for ? 5-containing nicotinic acetylcholine receptors (?5* nAChRs) in the habenulo-interpeduncular tract in regulating nicotine intake. We found that nicotine-induced activation of ? 5* nAChRs in the habenulo-interpeduncular tract triggered a negative motivational signal that served to reduce further nicotine consumption. Genetic variation in the CHRNA5-CHRNA3-CHRNB4 gene cluster, encoding the ? 5, ? 3 and ?4 nAChR subunits respectively, increases vulnerability to tobacco addiction and smoking- associated diseases including lung cancer. Here, we will fully define the role for ? 5*, ? 3* and ? 4* nAChRs in the MHb-IPN tract regulating nicotine reinforcement. Under Specific Aim I, we will assess nicotine self-administration behavior in knock-in mice in which the ? 5 nAChR subunit gene has been genetically modified to express a major risk allele for tobacco dependence in humans. Second, we will use an elegant combination of Double-floxed Inverted (DiO) adeno- associated viruses in Cre-expressing transgenic mice or in mice treated with Cre-expression virus to selectively re-express ? 5 nAChR subunits in discrete neuronal populations and pathways in the brains of ? 5 subunit knockout mice. We will then assess the impact on nicotine self-administration behavior in these rescued mice. Under Specific Aim II we will investigate the role for ? 3* nAChRs in the habenulo-interpeduncular tract in nicotine self-administration behavior using a combination of mouse behavioral genetics and virus-mediated gene transfer in mice. Should we find that ? 3 nAChR subunits in the habenulo-interpeduncular tract regulate nicotine intake in mice we will confirm these findings by assessing the effects of virus-mediated knockdown of this subunit in the habenulo-interpeduncular tract on nicotine self-administration in rats. Under Specific Aim III we will investigate the role for ? 4* nAChRs in the habenulo- interpeduncular tract in nicotine self-administration behavior using a combination of mouse behavioral genetics and virus-mediated gene transfer in mice. Should we find that ? 4 nAChR subunits in the habenulo-interpeduncular tract regulate nicotine intake in mice we will similarly confirm these findings by assessing the effects of virus-mediated knockdown of this subunit in the habenulo-interpeduncular tract on nicotine self-administration in rats. These studies promise to yield significantly new advances in our understanding of nicotine dependence and tobacco addiction. PUBLIC HEALTH RELEVANCE: Tobacco smoking results in greater than 5 million deaths each year. Even when using the most clinically efficacious smoking cessation agents available, approximately 80% of smokers attempting to quit will relapse within one year. This highlights our need to better understand neurobiology of the nicotine dependence, which drives tobacco addiction. During the previous funding period, we found that nicotinic acetylcholine receptors (nAChRs) containing ? 5 subunits (? 5* nAChRs) in the medial habenula (MHb) and interpeduncular nucleus (IPN) served to limit nicotine consumption, and are therefore protective against tobacco addiction. In this renewal application we will significantly extend these exciting findings. This program of research promises to identify new targets for the development of therapeutic agents for smoking cessation, and may therefore have a very significant positive impact on human health.